Herpes simplex viruses 1 and 2 (HSV-1, HSV-2) cause a wide range of diseases, including oral/genital lesions, corneal blindness and encephalitis. HSV-1 and HSV-2 can also be transmitted to newborns. Therapy for HSV relies primarily on nucleoside analogs such as acyclovir (ACV) that are insufficiently effective, and resistant viruses are becoming common. Therefore, there is an urgent need for better anti-herpesvirus drugs. The nucleotidyl transferase superfamily (NTS) of enzymes comprises a range of nucleases and recombinases that are essential for DNA replication, recombination, and nucleic acid turnover. They share key aspects of their enzymatic mechanisms. These similarities are great enough that many inhibitors of NTS enzymes cross-inhibit other NTS enzymes. This led us to hypothesize that HSV replication would be highly sensitive to inhibitors of NTS enzymes because such enzymes play many critical roles during viral DNA replication. Therefore, we screened 42 NTS enzyme inhibitors for anti-HSV-1 or -HSV-2 activity. We found: 1) Compounds from three different chemical classes profoundly inhibited both HSV-1 and HSV-2 with no toxicity in short-term assays; 2) the best inhibitors had 50% effective concentration values as low as 0.22 M; 3) five of these primary screening hits performed as well or better than the FDA-approved anti-herpesvirus drug ACV; 4) NTS inhibitors were effective against ACV-resistant HSV-1 and HSV-2; and 5) the inhibitors appear to target at least two phases of the viral replication cycle. In this R03 project, we will begin to define the frequency and patter with which HSV evolves resistance to inhibitors of NTS enzymes. Aim 1: Assess the frequency and pattern of resistance evolution to NTS enzyme inhibitors by HSV- 1. Classical viral drug-resistance evolution studies will be conducted to evaluate the propensity of the viruses to evade suppression by NTS inhibitors and to provide a collection of resistant viruses for genetic analyses. These data will help define whether NTS inhibitors would best be used alone or in combination therapies. Aim 2: We will sequence whole genomes from multiple independent lineages of HSV mutants resistant to NTS inhibitors to determine where mutations accumulate in response to compound treatment. These data will support follow-up efforts to address the mechanism of inhibition by implicating viral genes as possible drug targets. This R03 project is anticipated to guide development of anti-HSV drugs that could be used alone or in combination with the existing drugs to increase efficacy, reduce development of antiviral resistance, and provide salvage therapies for patients infected with drug-resistant viruses. They may have broader significance if, as is anticipated, NTS inhibitors prove to be active against other human pathogens in the herpesvirus family. Determine mutation patterns in genomes of viruses selected for resistance to NTS inhibitors